A nonsense mutation in IKBKB causes combined

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Aug 18, 2014 - (IKBKG) cause incontinentia pigmenti in heterozygous females and are ... males.10 Hypomorphic alleles are compatible with life in males, but ...
Blood First Edition Paper, prepublished online August 18, 2014; DOI 10.1182/blood-2014-04-571265

Full title: A nonsense mutation in IKBKB causes combined immunodeficiency Running title: IKBKB mutation causes Combined Immunodeficiency By: Talal Mousallem

1,2

2

3

*, Jialong Yang *, Thomas Urban , Hongxia Wang

2

2

2,5

3

Roberta E. Parrott , Joseph L. Roberts , David Goldstein , Rebecca H. Buckley Zhong

1

4

, Mehdi Adeli ,

2,5

, and Xiao-Ping

2,5

Department of Internal Medicine, Section of Pulmonary, Critical Care, Allergy and

Immunological Diseases, Winston-Salem, NC 27157, USA

2

Department of Pediatrics, Division of Allergy and Immunology, Duke University Medical Center,

Durham, NC 27710, USA

3

Center for Human Genome Variation, Duke University Medical Center, Durham, NC 27710,

USA

4

Hamad Medical Corporation, Doha, Qatar

4

Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou,

Guangdong 510515, China

5

Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA

*These authors contributed equally to this work.

Corresponding Author: Rebecca H. Buckley, M.D. Box 2898 or 362 Jones Building Duke University Medical Center Durham, North Carolina 27710 E-mail: [email protected] Telephone #: 919/684-3204 Fax #: 919/681-7979

Copyright © 2014 American Society of Hematology

Key Points:

1.

A nonsense mutation in IKBKB caused the absence of IKK

β and lack of T and B cell

activation through their antigen receptors.

2.

β is not necessary for development of T or B lymphocytes but is important for their

IKK

activation and development/function of NK cells.

2

Abstract Identification of the molecular etiologies of primary immunodeficiencies has led to

important insights into the development and function of the immune system. We report here

the cause of Combined Immunodeficiency in 4 patients from 2 different consanguineous Qatari

families with similar clinical and immunologic phenotypes. The patients presented at an early

age with fungal, viral and bacterial infections and hypogammaglobulinemia. Although their B-

and T-cell numbers were normal, they had low regulatory T-cell and NK-cell numbers.

Moreover, patients’ T-cells were mostly CD45RA

+

naïve cells and defective in activation

following TCR stimulation. All patients contained the same homozygous nonsense mutation in

IKBKB (R286X) revealed by whole-exome sequencing with undetectable IKKβ and severely

decreased NEMO proteins. Mutant IKK

β(R286X) was unable to complex with IKKα/NEMO.

Immortalized patient B-cells displayed impaired I

κBα phosphorylation and NFκB nuclear

translocation. These data indicate that mutated IKBKB is the likely cause of immunodeficiency

in these four patients.

3

Introduction

Mutations in genes important in T-cell or in both T- and B-cell development and function

cause severe combined immunodeficiency (SCID), with a majority of cases caused by mutations

in IL2RG, IL7RA, ADA, JAK3, RAG1, RAG2, or DCLRE1C.

1;2

In contrast to SCID, T- and B-cell

development is not as severely impaired in combined immunodeficiency (CID). CID has been

associated with hypomorphic mutations in SCID-causing genes as well as mutations in several

other genes: ZAP70, MHC class II deficiencies , PNP, ORAI-1, STIM1, NEMO, CARD11 and

MALT1;

3-7

however, the etiology of many CID patients remains unknown.

The I

κB kinase (IKK) complex contains 2 structurally related catalytic subunits, IKKα and

IKKβ and a regulatory subunit, IKKγ/NEMO.

8;9

Null alleles of the X-linked gene encoding NEMO

(IKBKG) cause incontinentia pigmenti in heterozygous females and are lethal in hemizygous

males.

10

Hypomorphic alleles are compatible with life in males, but cause immunodeficiency

and accompanying developmental abnormalities of teeth, hair, or sweat glands in many

patients.

3;11;12

Hypermorphic mutations in the gene encoding IkB

α cause autosomal-dominant

ectodermal dysplasia with T-cell immunodeficiency, undetectable memory T-cells, and absence

of response to CD3-TCR activation.

13

The IKK

α/β/NEMO complex, activated by antigen and

other receptors, phosphorylates the IκB molecules, leading to subsequent NF

κB nuclear

translocation to activate transcription of genes involved in immune responses.

14

We report a

homozygous nonsense mutation in IKBKB in four CID patients from 2 unrelated families and

compare and contrast our findings with those in two other recent reports of mutations in this

gene.

15;16

4

Patient, Materials and Methods:

Abbreviated information is presented here. Details are provided in the Supplemental

Material.

Patients

All studies were performed with the approval of the DUMC Institutional Review Board

and written informed consent of the patients’ parents in accordance with the Declaration of

Helsinki. The patients were members of 2 unrelated consanguineous families. Select clinical

features are presented in the Table with further details in the Supplemental Materials.

Immunologic Phenotype Analysis

Flow cytometry of peripheral blood leucocytes was performed with labeled antibodies.

Lymphocyte proliferation was assessed as previously described.

17

Exome sequencing, alignment and variant calling

Exome sequencing was performed in the Genomic Analysis Facility in the Center for

Human Genome Variation (Duke University). Sequencing libraries were prepared from DNA

extracted from patients’ leukocytes using the Illumina TruSeq library preparation kit following

the manufacturer’s protocol.

Establishment of EBV cell lines

EBV-transformed B-cell lines were established from peripheral blood leukocytes as

previously described.

18

5

Cloning of full-length and mutant HuIKK β, transfection and retroviral transduction

β ) or R286X mutant IKKβ cDNA, amplified from a WT human IKKβ

WT full-length (FL-hIKK

template (Addgene) with a forward primer 5’-GTGAACCGTCAGAATTGATCT-3’ and a reverse

primer 5’-gagtGtttaaacACATCATGAGGCCTGCTCCA-3’ or 5’-

CggaattcTCAGGGGTGCCACATCAGCATCA-3’, was cloned into the MIGR1 retroviral vector.

Retroviral vectors were transfected into the Phoenix-Ampho cells to generate amphotropic

retroviruses.

Cell stimulation, isolation of nuclear and cytoplasmic fractions, and immunoblot analysis

EBV-transformed B-cells, rested in DPBS at 37°C for 30 minutes, were stimulated with PMA (20

ng/mL) or anti-human CD40 (10 μg/mL, Biolegend) at 37°C for 10 or 20 minutes. Nuclear

extracts, cytosolic or total cell lysates were subjected to standard immunoblotting analysis.

Real-time quantitative PCR

Total RNA from PBMCs and EBV-transformed B-cells were used for real-time

quantitative PCR analysis.

Statistical analysis

Two-tail Student’s t-test was performed (*P < .05; **P < .01; ***P < .001).

Results and discussion:

All 4 infants were hypogammaglobulinemic (Table 1). Three demonstrated normal or elevated

numbers of T-cells, normal numbers of B-cells, but low numbers of switched memory B-cells

6

+

and NK-cells. Most T-cells were CD45RA . There were abnormally low numbers of CD45RO

+

cells and CD4 CD25

bright

+

+

or CD4 FOXP3

+

T-

T-regulatory-cells (Treg) (Supplemental Figure 1).

All patient T-cells displayed normal responses to PHA, but low responses to PWM or Con

A in 3 of them (Figure 1A). Strikingly, all patients’ T-cells failed to respond to candida or tetanus

toxoid antigens or anti-CD3 stimulation (Figure 1B and 1C).

Moreover, patients’ CD4 T-cells

were impaired for TCR-induced CD25 and CD69 upregulation (Figure 1D).

NK-cell function,

tested in one patient from each family, was impaired in both (data not shown).

WES on patients 1 and 2 representing the 2 unrelated families identified a single

candidate nonsense homozygous variant in IKBKB (R286X). The other two patients and the

parents carried the same homozygous and heterozygous variant, respectively (Supplemental

Figure 1C and 1D), which was absent in the 998 sequenced controls and in the exome variant

server.

Contrarily to healthy controls and a heterozygous sibling, anti-N- or -C-terminal IKK

antibodies failed to detect any full-length nor truncated IKK

β

β

in patients’ PBMCs and EBV-

transformed B-cell lines (Figure 1E and 1F), which was not caused by possible low quality of the

antibody (Supplemental Figure 1E).

Interestingly, NEMO was virtually undetectable but IKK

was not obviously altered in patient samples (Figure 1E and 1F). Both I

levels were considerably increased in patients’ PBMCs (Figure 1G).

κKβ

α

and NEMO mRNA

Contrarily to WT IKK

β,

β(R286X) could not associate with either IKK α or NEMO when overexpressed in Pheonix-eco

IKK

cells (Figure 1H).

7

Patients’

EBV-transformed

B-cells

displayed

reduced

degradation, and impaired nuclear accumulation of NF

which activates the PKC-IKK-NF

reconstitution

defective I

1F).

than

with

κBα/NFκB

full-length

κB

pathway (Figure 1I).

but

not

β (R286X),

IKK

κB

19

I

κBα

phosphorylation

and

following stimulation with PMA,

Such defects were corrected after

suggesting

that

β (R286X)

IKK

caused

activation in patient-derived B-cells (Figure 1J and supplemental figure

Concordantly, expansion of patient’s EBV-transformed B-cells, which was slower in vitro

their

sibling’s

or

parents’

counterparts,

could

reconstituted with full-length but not mutant IKK

β(R286X)

expressing IKK

β(R286X)

IKK

β

be

accelerated

to

WT

levels

when

(Figure 1J). Interestingly, patient B-cells

expanded slower than cells expressing GFP alone, suggesting that

exerted a dominant-negative function. Currently, it is unclear if IKK

interfere with the residual IKK

β(R286X) may

α/NFκB signaling in these cells.

Together, these observations indicate that IKK

β protein is absent in the patients and that the C-

β mediates its association with IKKα/NEMO, which may be important for the

terminus of IKK

stability of itself and NEMO, supporting that the nonsense mutation in IKBKB caused CID in

these patients. Our results indicate that IKK

β is not required for the development of T- and B-

cells and show a novel cause of CID related to defects in the NF

κB pathway. These findings

highlight the diversity of phenotypes associated with mutations in different components of the

NF

κB pathway.

dysplasia,

20

For example, most patients with mutations in IKBKG had ectodermal

which was absent in our IKK

β(R286X) patients even though they have almost

undetectable NEMO. Interestingly, while this manuscript was in preparation/review, two other

IKBKB mutations in CID patients were reported, one duplication of exon 13 (IKBKB13Dup)

8

15

and

the other Y107X nonsense mutation of IKBKB.

15;16

The clinical features of our patients were

similar to those in the other two reports, with early infections with candida, gram negative

bacteria, viruses and mycobacteria. The immunological features were also similar. All have

elevated numbers of naïve T cells that were poorly activated by antigens and anti-CD3; low

numbers of B-cells that were also naïve, and low numbers and function of NK-cells. There was a

paucity of CD45RO positive T cells, Tregs and

γ/δ T-cells. Similar to our study, mutant IKKβ

protein was undetectable and NEMO was decreased in IKBKB13Dup patients.

was not obviously decreased in our IKK

IKBKB13Dup patients.

not reported.

15

15

However, IKK

α

β(R286X) patients but was severely decreased in the β(Y107X) mutation on IKKα and NEMO expression was

The effect of IKK

16

Acknowledgements.

The authors thank the Cancer Center flow-cytometry core facility at Duke

University for cell sorting. We would like to acknowledge the following individuals for the

contributions of control samples: Dr. Gianpiero Cavalleri, Dr. Norman Delanty, Dr. Chantal

Depondt, and Dr. Sanjay Sisodiya; Dr. William B. Gallentine, Dr. Erin L. Heinzen, Dr. Aatif M.

Husain, Ms. Kristen N Linney, Dr. Rodney A. Radtke, Dr. Saurabh R. Sinha, and Ms. Nicole M.

Walley; Dr. Julie Hoover-Fong, Dr. Nara L. Sobreira and Dr. David Valle; Dr. William L. Lowe; Dr.

Scott M. Palmer; Dr. Zvi Farfel, Dr. Doron Lancet, and Dr. Elon Pras; Mr. Arthur Holden and Dr.

Elijah Behr; Dr. Annapurna Poduri; Dr. Patricia Lugar; Dr. Rasheed Gbadegesin and Dr. Michelle

Winn; Dr. Robert Brown; Dr. Gianpiero Cavalleri, Dr. Norman Delanty, Dr. Chantal Depondt; Dr.

Yong-Hui Jiang, Dr. Vandana Shashi and Ms. Kelly Schoch; Dr. Eli J. Holtzman; Dr. Sarah Kerns

and Dr. Harriet Oster; Dr. Doug Marchuk; Dr. Demetre Daskalakis; Dr. Nicole Calakos; Dr. Francis

J. McMahon and Nirmala Akula; Dr. M Chiara Manzini.

9

Supported by the NHLBI GO Exome Sequencing Project (HL-102923, HL-102924, HL-102925, HL-

102926 and HL-103010) and by NIAID (AI076357, AI079088, and AI101206 to X-P.Z.) The

collection of control samples and the production of sequence data were funded in part by The

Epilepsy Phenome/Genome Project U01NS053998; Epi4K Project 1 – Epileptic Encephalopathies

U01NS077364; Epi4K Sequencing, Biostatistics and Bioinformatics Core U01NS077303; NIAID

Grant UO1AIO67854 (Center for HIV/AIDS Vaccine Immunology ("CHAVI")), and an award from

Biogen Idec.

Conflict of Interest Statements. None of the authors has any conflict of interest to declare. Authorship Contributions. TM designed the study, analyzed sequencing data, wrote the paper; JY designed and performed functional studies, contributed to manuscript preparation; TU

analyzed the sequencing data; HW participated in functional studies; MA referred the patients,

conducted immunologic investigations; REP performed extensive immunologic studies,

contributed to manuscript preparation; JLR contributed to data analysis and manuscript

composition; DG oversaw the whole genome sequencing and data analysis, contributed to

manuscript; RHB designed the study, wrote the paper, performed extensive immunologic

evaluations; XPZ designed functional studies, analyzed data, and wrote the paper.

10

Reference List

1. Fischer A, Le Deist F, Hacein-Bey-Abina S et al. Severe combined immunodeficiency. A model disease for molecular immunology and therapy. Immunol.Rev. 2005;203:98-109. 2. Buckley RH. Transplantation of hematopoietic stem cells in human severe combined immunodeficiency: longterm outcomes. Immunol.Res. 2011;49(1-3):25-43. 3. Orange JS, Brodeur SR, Jain A et al. Deficient natural killer cell cytotoxicity in patients with IKKgamma/NEMO mutations. J.Clin.Invest 2002;109(11):1501-1509. 4. Feske S, Gwack Y, Prakriya M et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature. 2006;441(7090):179-185. 5. Picard C, McCarl CA, Papolos A et al. STIM1 mutation associated with a syndrome of immunodeficiency and autoimmunity. N.Engl.J.Med. 2009;360(19):1971-1980. 6. Jabara HH, Ohsumi T, Chou J et al. A homozygous mucosa-associated lymphoid tissue 1 (MALT1) mutation in a family with combined immunodeficiency. J.Allergy Clin.Immunol. 2013;132(1):151158. 7. Stepensky P, Keller B, Buchta M et al. Deficiency of caspase recruitment domain family, member 11 (CARD11), causes profound combined immunodeficiency in human subjects. J Allergy Clin Immunol 2013;131(2):477-485. 8. Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M. The IκB kinase complex (IKK) contains two kinase subunits, IKKα and IKKβ, necessary for IκB phosphorylation and NF-κB activation. Cell 1997;91(2):243-252. 11

9. Courtois G, Israel A. IKK regulation and human genetics. Curr.Top.Microbiol.Immunol. 2011;349:73-95. 10. Smahi A, Courtois G, Vabres P et al. Genomic rearrangement in NEMO impairs NF-κB activation and is a cause of incontinentia pigmenti. The International Incontinentia Pigmenti (IP) Consortium. Nature. 2000;405(6785):466-472. 11. Niehues T, Reichenbach J, Neubert J et al. Nuclear factor κB essential modulator-deficient child with immunodeficiency yet without anhidrotic ectodermal dysplasia. J Allergy Clin Immunol. 2004;114(6):1456-1462. 12. Orange JS, Levy O, Brodeur SR et al. Human nuclear factor κ B essential modulator mutation can result in immunodeficiency without ectodermal dysplasia. J.Allergy Clin.Immunol. 2004;114(3):650-656. 13. Courtois G, Smahi A, Reichenbach J et al. A hypermorphic IκBα mutation is associated with autosomal dominant anhidrotic ectodermal dysplasia and T cell immunodeficiency. J Clin Invest. 2003;112(7):1108-1115. 14. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu.Rev.Immunol. 2000;18:621-663. 15. Pannicke U, Baumann B, Fuchs S et al. Deficiency of innate and acquired immunity caused by an IKBKB mutation. N.Engl.J Med. 2013;369(26):2504-2514. 16. Burns SO, Plagnol V, Gutierrez BM et al. Immunodeficiency and disseminated mycobacterial infection associated with homozygous nonsense mutation of IKKβ. J Allergy Clin Immunol. 2014 Jul;134(1):215-218.e3. 12

17. Buckley RH, Schiff SE, Sampson HA et al. Development of immunity in human severe primary T cell deficiency following haploidentical bone marrow stem cell transplantation. J Immunol. 1986;136(7):2398-2407. 18. Sugden B, Mark W. Clonal transformation of adult human leukocytes by Epstein-Barr virus. J Virol. 1977;23(3):503-508. 19. Krishna S, Zhong X. Role of diacylglycerol kinases in T cell development and function. Crit Rev.Immunol. 2013;33(2):97-118. 20. Hanson EP, Monaco-Shawver L, Solt LA et al. Hypomorphic nuclear factor-κB essential modulator mutation database and reconstitution system identifies phenotypic and immunologic diversity. J.Allergy Clin.Immunol. 2008;122(6):1169-1177. 21. Shearer WT, Rosenblatt HM, Gelman RS et al. Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric AIDS Clinical Trials Group P1009 study. J.Allergy Clin.Immunol. 2003;112(5):973-980.

13

Table 1: Clinical Features, Immunoglobulins and Lymphocyte Subsets Family 1 Patient 1

Patient 2

Family 2 Patient 3

Patient 4

Sex

Female

Male

Female

Male

Age at Presentation

5 mo.

11 mo.

7 mo.

6 mo.

Infections

Candida,

Candida,

Candida

BCG,

rotavirus,

Klebsiella,

BCG

CMV

Parameter

Normal Range

Clinical Features

Treatment

Candida,

Ablated, HLA-

Ablated,

Ablated, T

Ablated, T

identical

unrelated

cell depleted

cell

paternal

donor cord

paternal

depleted

marrow HSCT

blood HSCT

marrow

maternal

HSCT

marrow HSCT

Outcome

Alive and well

Died 2 mos.

Died 1 yr.

Died 13

later

later

mo. later

Immunoglobulins*

1

IgG

82

712

367

173

192-515

IgA

low

0

0

0

12-31

IgM

low

0

0

22

39-92

11,550(96)

14,792(96)

6,861(81)

9,118(82)

2,940-4,560

Flow Cytometry∞ T and NK Cells CD3

(49-76) CD4

8,656(72)

12,380(81)

5,946(70)

7,498(67)

1,860-3,360 (31-56)

CD8

2,881(24)

2,458(16)

1,262(15)

1,888(17)

11,118(93)

14,792(96)

6,852(81)

9,062(81)

α/β

11,502(96)

14,561(95)

6,683(79)

9,040(81)

P

3,000-4,680 (50-78)

Ti

P

720-1,440 (12-24)

CD28

P

Q

3,480-5,040 (58-84)

Q

γ/δ

180(2)

15(0)

42(1)

939(8)

0-540 (0-9)

CD16

120(1)

154(1)

644(8)

335(3)

180-1,080

Ti

CD4,CD25

bright

(3-18) 240(2)

ND

158(1.6)

ND

Q

24-336 (0.4-5.6)

Q

B Cells CD19

384(3)

461(3)

14

881(10)

894(8)

840-2,220

Q

(14-37) CD20

312(3)

476(3)

813(10)

950(9)

300-1,020 (5-17)

Switched Memory

2(13.3)

ND

2(9.2)

ND

B

P

Q

1-224 (22.4-79.4)

Q

Activation CD45

11,994(100)

15,345(100)

8,428(100)

10,526(94)

5,520-6,000 (92-100)

CD45RO,CD3

1063(9)

59(0)

521(8)

27(0)

177-1,140 (6-25)

CD45RA,CD3

9783(85)

14,274(97)

6,360(93)

8,589(94)

P

441-2,006 (15-44)

CD45RA,CD3,CD62L

8697(75.3)

ND

6,237(90.9)

ND

Q

Q

338-2,513 (11.5-55.1)

1 *Values are expressed as mg/dl. The patient was receiving IG replacement. ∞Values are expressed as cells/mm3 or (percentage of lymphocytes).

P

Normal values are the 95%

confidence intervals for ninety 6-12 mo-old healthy control subjects.

21 Q

Normal values are the

95% confidence intervals for 2338 healthy adult control subjects from the authors’ laboratory.

15

Q

Legend to Figure Figure 1. Contribution of IKKβ (R286X) mutation to CID. (A-C) Patients’ blood lymphocytes responded normally when stimulated with PHA but had variably low responses to Con A and

PWM (A). However, they failed to respond when stimulated with candida and tetanus antigens

(B) or soluble or immobilized anti-CD3 (C).

(D) Impaired upregulation of T cell activation

markers in patients’ CD4 T cells following overnight stimulation with plate-bound anti-CD3 or

PHA. (

E,F) Neither full-length nor truncated mutant IKKβ(R286X) protein is detectable in

patients (PT), siblings, and normal PBMC (E) and EBV-transformed B-cells (F) by immunoblotting

analysis with anti-N- and –C-terminal IKK

β antibodies. (G) mRNA levels of IKKα, IKKβ, and

NEMO in PMCs detected by real-time qPCR. (

IKK

α/NEMO.

H) IKKβ (R286X) is not able to form a complex with

Cell lysates from Phoenix-Eco cells transfected with either Flag-tagged-FL-IKK

β,

β(R286X), or vector control were subjected to immunoblotting analysis directly

Flag-tagged-IKK

(left panels) or after immunoprecipitation with anti-Flag antibody conjugated agarose beads

I

(right panels). ( ) Defective I

κBα/NFκB signaling in patient-derived B-cells can be reverted by

full-length (long) but not mutant IKK

β(R286X).

EBV-transformed B-cells of a sibling and patients

stably infected with retrovirus expressing WT IKK

β or with control vector were rested in PBS at

o

37 C for 30’ followed by PMA stimulation for 10 and 20 minutes. Immunoblotting analysis of

J

cytosolic fractions (top panel) or nuclear extracts (bottom panel) with indicated antibodies. ( )

Decreased expansion of patient-derived B-cells can be corrected by full-length but not mutant

β.

IKK

*P < .05; **P < .01; ***P < .001 determined by Student’s T-test.

16

Figure 1

15 10

8

C

Medium Candida Tetanus

6

15 CPM (x104)

20

4

Medium Anti-CD3 Immob anti-CD3

10

5

2 0 PT1

PT2

D

PT3

PT1

unsti

PT1

PT2

E WT

PT4

PT1

NC

PT3

% Max

80

60

60

40

40

20 0

35

20 0 102

103

104

105

0

CD69

0 102

103

104

105

IKK-β (N-ter)

CD25

IKK-β (C-ter)

H

G NC

IKK-β (C-ter) IKK-α NEMO β-actin

35 β-actin

PT+WT PT+vector sibling 0 10 20 0 10 20 0 10 20

*** flag

** 3.0

2.0

IKK-α

IKK-β

35

NEMO

IKK-α NEMO

J

sibling PT+WT PT+vector 0 10 20 0 10 20 0 10 20

Cell number (x104)

p-IκBα β-actin

100

35 IKK-α NEMO β-actin

***

IκBα

histone H3

flag

***

IKK-β

NF-κB p65

100

***

0.0

I

PT3

PT1

Sibling

6.0 Relative mRNA level

NC 100

sibling PT

NC

sibling PT

F IKK-β (N-ter)

NC

WT-IKKβ IKKβ(R286X) Vector

100

80

Vector

100

WT-IKKβ IKKβ(R286X)

PHA

PT4

IKK-α NEMO β-actin

100

PT3 anti-CD3

PT2

NC

0

NC

Sibling

PT4

PT3

PT3

PT1

PT2

NC

PT1

PT1 PT3 Sibling

0

NC

5

Sibling

CPM (x104)

B

Medium PHA Con A PWM

25

CPM (x104)

A

20

*** ***

15

**

10 5 0

PT + WT PT + mutant PT + vector

** ** * *

24h

48h

sibling PT

72h